There have been hitherto known various immunoassay methods as the method for detecting a trace component in the research fields such as medical diagnosis, clinical examinations, etc., and concerning this, various techniques have been proposed about reagents labelled with dyes, assay methods or devices.
1) Labelling reagent
For labelling reagents, there have been developed antigens or antibodies labelled with radioisotopes, luminous reagents or enzymes.
Among them, those having the highest sensitivity are those labelled with radioisotopes, but they cannot easily be handled. On the other hand, those labelled with enzymes are effective only for a part of substance. For this reason, sensitization of reagents labelled with luminous materials has been desired.
As such a high sensitivity reagent which can be used for luminous immunoassay, in Japanese Unexamined Patent Publication No. 61468/1983, an immunological reagent comprising an organic polymer having a plural number of luminescents bound to either one of an antibody or an antigen and an immunoassay by use thereof are proposed, and also, in U.S. Pat. No. 4,166,105, a detecting reagent of an analyte which is the primary reactant (antibody) capable of specifically reacting with a polymeric backbone possesing reactive functional group analyte, and has a large number of fluorophor molecules bound thereto is proposed, respectively. However, these reagents are not sufficient in the amount of the dye which can be bound. Therefore when an insensitive fluorescent dye excited at a long wavelength line is used, there is involved the problem that detectable sensitivity is not practical.
Also, in Japanese Unexamined Patent Publication No. 252265/1985, there is disclosed an assay method of a biologically active substance by using a reagent wherein a water-soluble organic polymer bound with a luminous reagent is bound to avidin. However, this reagent and method may probably reduce immunological activity because an antibody or an antigen is bound to avidin through biotin with small molecular weight, whereby a large amount of biotin is bound around the antibody or the antigen. Therefore it is necessary to make a treatment for prevention of this, and also there is the problem that a water-soluble polymer can be bound to avidin with difficulty due to steric hindrance.
2) Fiber optics used for luminous immunoassay
In luminous immunoassay, the method of exciting a fluorescent dye (luminous reagent) or transmitting fluorescence by utilizing a fiber optics is effective, and various techniques have been disclosed.
For using a fiber optics as the sensor, it is necessary to immobilize an antigen or an antibody onto a fiber optics, and as the techniques for this purpose, there are the membrane immobilization method which immobilize an antigen or an antibody onto a film such as cellophane, the entrapping method which seals an antigen or an antibody within pores of an acrylamide gel, etc. The former method has the problem of lowering sensitivity due to light scattering, and the latter has the problem of poor responsiveness. For such reasons, as the method for covalently bonding an antigen or an antibody directly to a fiber optics, in Analytical Chemistry, Vol. 59, No. 8, pp. 1226-1230, there is disclosed a method in which (3-glycidoxypropyl)trimethoxysilane (GOPS) which is a silane coupling reagent is allowed to react with the silanol groups on the surface of quartz fiber optics, subsequently this is treated with HIO.sub.4 to introduce formyl groups onto the surface of the quartz fiber optics, and allowing amino groups of a protein with the formyl groups to effect immobilizing. However, this method is an effective only for quartz fiber, and cannot be used for plastic fiber optics. Plastic fiber optics are inexpensive in price, can be easily polished, are flexible and easy in handling, and hence it has been desired to have a method to bind a protein such as an antigen or an antibody to a plastic fiber.
Also, as the device using fiber optics, Japanese Unexamined Patent Publication No. 501873/1984 (U.S. Pat. No. 4,582,809) discloses an instrument for immunoassay and method, and Japanese Unexamined Patent Publications No. 123358/1987 and No. 501102/1987 (Switzerland Patent Application No. 5306/84-5) disclose fiber optic immunosensor, respectively. However, in these specifications, there is no description about the technique of labelling an antigen or an antibody highly sensitively, and for this reason, they can be utilized only for the case when a highly sensitive fluorescent dye as excited by Hg lamp, Xe lamp or Ar laser is employed as light source, and also there is the problem that the device is large in scale and expensive.
3) Assay method
There have also been made various proposals about the methods of immunoassay using luminous reagents. For example, in Japanese Unexamined Patent Publication No. 24450/1985, there is proposed a method for assaying biomaterials which measures the luminous intensity by the luminescence reaction of a luminescent binding avidin which is bound to an immuno-complex through biotin-avidin interaction. This method has avidin-biotin interaction interposed between the luminous reagent and an antibody or an antigen, but no other organic polymer interposed therebetween. Therefore, the amount capable of attaching luminescents is small, whereby there is the problem that insensitive luminescents can not be used. Also, as the reagent using biotin-avidin interaction, a labelled immunological material is proposed in Japanese Unexamined Patent Publication No. 30667/1983 (Switzerland Patent Application No. 6989/8101). However, this technique has the problem that the immunological material is labelled with an enzyme, and the treatment for measurement of enzyme activity is cumbersome.